1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the invention relates to methods and compositions for modifying plant biosynthetic pathways.
2. Description of the Related Art
Plants are capable of synthesizing a large variety of low molecular weight organic compounds, which are collectively called secondary metabolites. In contrast to primary metabolites that are common to all plants, many secondary metabolites are differentially distributed among limited taxonomic groups within the plant kingdom. Plant secondary metabolites play important roles in plant-environment interactions, and in human nutrition and medicine (DellaPenna 1999; Dixon, 2001; La Camera et al., 2004). Secondary metabolites usually exist in low abundance in plants. Because of the structural complexity of secondary metabolites, their chemical synthesis is not only difficult and expensive, but also often results in low yields. It is therefore desirable to be able to manipulate biosynthetic pathways for large-scale production of targeted secondary metabolites in plants, such as by enhancing the expression of endogenous genes or by introducing foreign genes (Dixon, 2005; Galili and Hofgen, 2002; Thelen and Ohlrogge 2002).
Phenylpropanoids are one of the largest groups of plant secondary metabolites, and are synthesized from the aromatic amino acid L-phenylalanine. Isoflavonoids are derived from the phenylpropanoid pathway and are distributed predominantly in the Leguminosae. They were initially recognized for their roles in plant disease resistance and induction of nodulation (Dixon, 2001). They have also received much attention in recent years due to their estrogenic, antioxidant, and anticancer activities in humans (Cornwell et al., 2004; Dixon and Ferreira, 2002; Dixon, 2004). It is desirable, in the long term, to be able to produce isoflavonoids in a wide range of plants and crops besides legumes for dietary disease prevention.
In nature, enzymes have often evolved as multi-domain proteins in order to perform tasks that require more than one function. Nature's strategy has been adopted by scientists from different disciplines, in particular E. coli and yeast researchers, to develop recombinant multifunctional proteins (Bülow, 1990; James and Viola, 2002; Nixon et al., 1998). However, this approach has not yet been broadly applicable to plants (Tian and Dixon, 2006), whose secondary metabolism presents a more complicated biochemical context than yeast or E. coli. An in-frame fusion of thiolase and reductase genes was recently constructed for polyhydroxybutyrate biosynthesis in Arabidopsis (Kourtz et al., 2005). The fusion protein exhibited thiolase and reductase activities in E. coli, though plants transformed with this construct produced less polyhydroxybutyrate than plants expressing thiolase and reductase individually.
While the foregoing studies have provided a further understanding of the biosynthesis of plant secondary metabolites, methods for the efficient modification of most secondary metabolites have been lacking. This has been particularly true in the case of isoflavonoid biosynthesis. There, therefore, remains a great need in the art for the development of methods and compositions that would increase the efficiency by which isoflavonoid biosynthesis can be modified in plants.